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Iraqi National Journal of Chemistry,2011,volume 44
‫ المجلد الرابع واالربعون‬2011-‫المجلة العراقية الوطنية لعلوم الكيمياء‬
An Indirect Spectrophotometric Determination of Mesna in
Pharmaceuticals and Environmental Samples
Nief Rahman Ahmed
Department of Environmental Technology,Colleg of Environmental, University of
Mosul
(NJC)
(Recevied on 24/5/2011 )
(Accepted for publication
31/10/2011)
Abstract
Two indirect spectrophotometric methods were proposed for the determination
of mesna in pure and pharmaceutical preparations using sodium hypochlorite and two
indicators, methyl orange and congo red. The methods involved addition of a known
excess of sodium hypochlorite to mesna in acidic medium, followed by determination
of residual sodium hypochlorite by reacting with a fixed amount of either methyl
orange, measuring the absorbance at 505nm (method A) or congo red measuring the
absorbance at 605nm (method B). In both methods the amount of sodium
hypochlorite reacted correspond to the amount of mesna and the measured absorbance
was found to increase linearly with the concentration of mesna, which is corroborated
by the correlation coefficient of 0.9996 and 0.9998 for method A and B respectively.
The two methods obey Beer’s law for 0.08-1.6 µg mL -1 and 0.2-2.0 µg mL-1 for
method A and B respectively. The apparent molar absorptivities were calculated to be
1.02×104 and 1.5×103 L mol-1 cm-1 for method A and method B respectively. The
limit of detection (LOD) and quantification (LOQ) were calculated to be 0.016
and0.05µg mL-1 for method A and 0.048 and 0.15 µg mL-1 for method B. The intraday and inter-day precision and accuracy of the two methods were evaluated
according to the current ICH guidelines. Both methods of comparable
accuracy(relative error) better than ± 1.8%. and precision of < 2%. The proposed
method was proved to be selective and applied to the determination of mesna in
pharmaceutical preparations and environmental water samples.
Keywords: Mesna, Spectrophotometry, Pharmaceutical preparations,Environmental
samples.
‫الخالصة‬
‫تم اختيار طريقتين طيفيتين غير مباشرتين لتقدير الدواء السرطاني مزنا بحالته النقية وفي بعض‬
‫مستحضراته الصيدالنية باستخدام هايبوكلورات الصوديوم وكاشفين هما المثيل البرتقالي و الكونكو االحمر‬
‫تتضمن الطريقة اضافة زيادة معلومة من هايبوكلورات الصوديوم الى الدواء مزنا في الوسط الحامضي ثم قياس‬
‫ نانوميتر‬505 ‫ حيث يقاس عند‬.‫المتبقي من هايبوكلورات الصوديوم بمفاعلته مع كمية ثابتة من المثيل البرتقالي‬
492
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‫ المجلد الرابع واالربعون‬2011-‫المجلة العراقية الوطنية لعلوم الكيمياء‬
‫ وفي كلتا الطريقتين‬.)‫ نانوميتر ( الطريقة الثانية‬605 ‫( الطريقة االولى ) او الكونكو األحمر حيث يقاس عند‬
‫فإن كمية هايبوكل ورات الصوديوم المتفاعلة تمثل كمية مزنا وان االمتصاص سوف يزداد مع زيادة تركيز مزنا‬
.‫ للطريقة األولى والثانية على التوالي‬0.9998‫ و‬0.9996 ‫والذي يمثل باستقامة الخط المستقيم ڊ‬
0.2-2.0 ‫ جزء بالمليون و‬0.08-1.6 ‫وفي كلتا الطريقتين فإن قانون بير يسري على الكميات التي تتراوح بين‬
.‫ لتر‬1.5×103 ‫ و‬1.2×104 ‫جزء بالمليون لكلتا الطريقتين على التوالي وان معامل االمتصاص الموالري هو‬
0.05 ‫ و‬0.016 ‫ وان حد الكشف والحد الكمي تم ايجاده‬.‫ لكلتا الطريقتين على التوالي‬1-‫ سم‬.1-‫مول‬
.‫مل للطريقة الثانية‬/‫ مايكرو غرام‬0.15 ‫ و‬0.048 ‫مايكروغرام لكل مللتر للطريقة االولى و‬
‫ فكلتا الطريقتين لهما‬ICH ‫ان ضبط الطريقتين خالل اليوم الواحد او بين يوم وآخر و تقيمهما اعتماداً على دليل‬
‫ وقد تم دراسة المواد المتداخلة مع مزنا والتي عادة‬. %2 ‫ وضبطهما اقل من‬% 1.6 ‫دقة) خطأ نسبي) اقل من‬
‫تتواجد معها ووجد بأنها ال تسبب أي تداخل وكلتا الطريقتين طبقت بنجاح لتقدير مزنا في مستحضراتة‬
.‫الصيدالنية و في النماذج البيئية‬
monitored
for
hoematuria
and
proteinuria throughout the treatment
period. However, frequent emptying of
the bladder should be avoided. recently
mesna used as antioxidant agent
against
acetaminophen
toxicity(4)
.Mesna is rapidly oxidized to dimesna,
so in pharmaceutical formulation it
must be stabilized using EDTA,
sodium hydroxide and inert gas
atmosphere. The reducing character of
mesna should be taken into account in
the design of any analytical methods(5)
The literatures revealed that mesna has
been determined by means of a few
analytical methods. These include:
HPLC(5-8).Chemiluminescence-flow
injection analysis(9) .B.P method
described a titrimetric assay for pure
drug only(10). The present work is to
provide
a
simple,
sensitive
spectrophotometric method for the
determination of mesna in dosage
forms and water samples, utilize
sodium hypochlorite and two dyes
methyl orange and congo red.
Introduction
Mesna, (sodium 2-sulfanylethane
sulfonate) with a molecular formula
of C2H5NaO3S2
and molecular
weight of (164.18 g mol-1). Its
structural formula is as follows:
HS-CH2-CH2-SO3-Na+
A nucleophilic thiol doner often used
for the prevention of urothelial toxicity
in
patients
treated
with
the
antineoplastics
ifosfamide
or
cyclophosphamid. In the kidney,
dimesna, the inactive metabolite of
mesna is redused to free mesna which
has thiol groups that react with the
metabolitied of ifosfamide and
cyclophophamide, including acrolein,
considered to be responsible for the
toxic effect on the bladder(1-3).
The aim of mesna therapy is to ensure
adequate levels of mesna in the urine
throughout the period during which
these toxic metabolites, are present and
the duration of mesna treatment should
therefore
equal
that
of
the
antineoplastic treatment. Plus the time
taken for the concentration of
antineoplastic metablites in the urine to
fall to non-toxic levels. Urinary output
should be maintained and the urine
Experimental
1 Apparatus
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Optima SP 3000 plus UV-Visible
spectrophotometer (Optima .Japan)
with 1.0 cm quartz cells was used for
the absorbance measurements.
‫ المجلد الرابع واالربعون‬2011-‫المجلة العراقية الوطنية لعلوم الكيمياء‬
solution were added. The content was
mixed and let stand for 5 min with
occasional shaking. Finally, 1mL of
0.1% methyl orange solution was
added and the volume was diluted to
the mark with distilled water and
mixed well. The absorbance of each
solution was measured at 505 nm
against a reagent blank .
2 Reagents
All chemicals used were of analytical
purity grade and all solutions were
prepared with distilled water. A
standard sodium hypochlorite solution
(0.1%) was prepared by dilution of
1.25mL of 8% sodium hypochlorite to
100 mL with distilled water, store in a
dark
bottle
and
standardized
iodometrically every 4-5 days. This
solution stored in a dark bottle(11).
Congo red: 0.1 % was prepared by
dissolving 0.1 g accurately weighed
dye in water, and diluting to 100 mL
in volumetric flask.
Methyl orange 0.1% was prepared by
dissolving 0.1 g accurately weighed
dye in water and diluting it to 100 mL
in volumetric flask.
Standard material and pharmaceutical
preparations (mesnan tablet 400
mg,and mesnan 100mg injection ) were
provided
from state company of
drug industries and medical appliance
(NDI) Ninavah- Iraq .
Standard
solution
of
mesna
-1
(100µg ml ).
This solution was prepared by
dissolving 0.1 g of pure drug in 1L
distilled water. It was later diluted with
water to get concentration of
10 µg ml-1.
Method B:Different aliquots of standard solution
equivalent to 5-50 µg of mesna
solution were transferred into a series
of 25 mL volumetric flask, 1mL of
HCL and 3 mL of sodium hypochlorite
were added. The content was mixed
well and the flasks were kept aside for
5 min with intermitten shaking.
Finally, 3mL of 1% congo red solution
was added to each flask, the volume
was diluted to the mark with distilled
water, mixed well and absorbance was
measured against a reagent blank at
605nm.
4 Preparation of Mesna drugs
1 Tablets
To minimize a possible variation in the
composition of the tablets, the mixed
content of 20 tablets, were weighed
and grounded, then the powder
equivalent to 100 mg of mesna was
stirred well with water for 15min and
the volume was made to 1L with
distilled water ,filtered through
whatman No. 42 filter paper and 10
mL of this solution was diluted to 100
ml by distilled water and aliquot of this
solution was treated as described above
for analytical procedure.
3 Analytical procedures
Method A:Different aliquots of standard mesna
solution equivalent 2-40 µg were
transferred into a series of 25mL
volumetric flasks, 1mL of 1N HCL,
and 5 ml of sodium hypochlorite
2 Injection
1ml vail containing 100 mg of mesna
was transferred into 1L volumetric
flask and diluted up to the mark with
distilled water, 10mL of this solution
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was diluted to 100mL with distilled
water and a aliquot of this solution was
treated as described above for
analytical procedure.
Di mesna
+ Un reacted NaOCL
3 Preparation
samples
Un reacted NaOCL +Methyl
orange → Bleached methyl orange
(measured at505nm)
of
spiked
Method A
water
To
demonstrate
the
practical
applicability of the proposed method,
real water samples were analysed by
this method. Drinking water from the
mosul water-supply net work ,
industrial waste water from the state
company for drug industries and
medical appliances Mosul-Iraq, were
spiked with the concentrations ranging
from 0.1-1.5mg/ml of mesna and a
liquot of this solution was treated as
described
above
for
analytical
procedure.
Method B
Un reacted NaOCL + Congo red→
Bleached congo red (measured at
605nm)
When
added
in
increasing
concentration to a fixed concentration
of sodium hypochlorite, mesna
consumes the latter proporationally and
there is a concomitant drop in the
remaining concentration of sodium
hypochlorite. When a fixed indicator
concentration is added to decreasing
concentration of sodium hypochlorite,
a concomitant increase in the indicator
concentration result a proportional
increase in absorbance at the respective
‫ ג‬max is observed with increasing
concentration of mesna.Fig (1and 2).
Result and Discussion
The versatility of sodium hypochlorite
as an analytical reagent can be gauged
by
its
applications
in
the
spectrophotometric determination of
many
organic
compounds
of
therapeutic importance(12-16).. The use
depends mainly on its ability to effect
the oxidation of diverse functional
groups. Taking advantage of the rapid
oxidation
reaction
of
sodium
hypochlorite with mesna.Mesna is a
reducing agent owing to the presence
of thiol group(-SH) in its structure.
The proposed spectrophotometric
methods are indirect and based on the
determination of residual sodium
hypochlorite after bringing the reaction
between
mesna
and
sodium
hypochlorite to completion. The
residual sodium hypochlorite was
determined by
methyl orange or
congo red indicators.
Fig[1]:Absorption spectra of method
A; a ,reagent blank against
water.,b,c and d after addition of
10,30 and 50µg of mesna.
2 HS-CH2-CH2-SO3Na + NaOCL
(Known excess) → NaO3-CH2-CH2S-CH2-CH2-SO3Na
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recommended procedure for both
methods. The reaction between mesna
and sodium hypochlorite and the
determination of the latter by reacting
with the indicators. HCL medium
(1mL of 1N) was found to be ideally
suited. Reaction time of 5mint is not
critical and any delay up to 6h did not
affect the absorbance reading.
3-1 Method Validation
Under the optimized conditions,
A liner correlation was found between
absorbance at ‫ ג‬max and mesna
concentration.Fig (3 and 4).
A = 0.016 + 0.303 C
R
=
0.996
n = 6 (method
A)
A = 0.0018 + 0.091C
R
=
0.998
n = 6 (method
B)
Where A is the absorbance and C is
concentration in µg.ml-1, R is the
regression coefficient and n is the
number of concentration levels.
Fig[2]:Absorption
spectra
of
methodB ; a ,reagent blank against
water.,b,c and d after addition of
10,30 and 50µg of mesna.
Preliminary
experiments
were
preformed
to
fix
the
upper
concentration of the indicator that
could
be
determined
spectrophotometrically and this was
found to be 1 and 3mL of 0.1% of
methyl orange and congo red
respectively. A sodium hypochlorite
concentration 500 µg was found to
bleach the red color due to 100 µg of
methyl orange where as 300µg was
required to destroy the blue color due
to 300µg congo red. Hence, different
concentration of mesna were reacted
with hypochlorite solution 5 ml of
0.1% method A and 3mL of 0.1%
sodium hypochlorite in method B,
followed by determination of residual
sodium hypochlorite under the
Fig[3]: Calibration curve of mesna with
methyl orange.
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Fig[4]:Calibration curve of mesna with
congo red
Table 1:Method validation of the spectrophotometric determination of mesna.
Parameter
Method A
Method B
‫ ג‬max (nm)
505
605
-1
Beer’s law limit (µg mL )
0.08-1.6
0.2-2
-1
-1
4
Molar absorptivity (L mol cm )
1.02×10
1.5×103
Limit of detection (µg mL-1)
0.016
0.05
Limit of quantification
0.048
0.15
Intercept
0.016
0.0018
Slope
0.303
0.091
Correlation coefficients ,(r)
0.996
0.998
The optical characteristics such as
Beer’s Law, limits and molar
absorptivity values is given in Table
(1). The limit of detection (LOD) and
quantication
(LOQ)
calculated
according to ICH guidelines (17) as the
ratio of 3.3 and 10 standard deviations
of the blank (n = 6) and the slope of
the calibration line.
deviation (%) values are summarized
in table (2). From Table (II), it is clear
that method A with a relative error of
1.3% is as accurate as method B with
1.6%. Moreover, both methods are
found to be precise with RSD values <
2%. For a better picture of
reproducibility on a day-to-day basis, a
series of experiments were performed,
in which the standard drug solution
was determined at three different levels
each day for six days, with all solutions
being prepared a fresh each day. The
day-to-day relative standard deviation
values were in the range of 1.1-1.8 %
and represent the best appraisal of
repeatability of the proposed methods.
3-2 Accuracy and Ruggedness of the
proposed methods
To evaluate the accuracy and intra-day
precision of the methods a pure drug
solution was analysed at two different
concentrations, each determination
being repeated six times. The relative
error (%) and relative standard
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‫ المجلد الرابع واالربعون‬2011-‫المجلة العراقية الوطنية لعلوم الكيمياء‬
Iraqi National Journal of Chemistry,2011,volume 44
Table ( 2) Accuracy and precision of the proposed methods.
Method
Mesna conc. taken µg Conc.found Er (%)a
RSD(%)
( µg )
A
20
20.22
1.1
1.3
40
40.52
1.3
1.8
B
10
10.12
1.2
1.5
30
30.48
1.6
1.1
a: Mean of six determinations
3-3 Interference studies
In order to assess the possible
applications of the two proposed
methods, the effect of substances that
often accompany with mesna in
various
pharmaceutical
products
(Tablets and injections) were studied
by adding different amount of
interferents to 20 µg of mesna. An
attractive featuer of the
method is its relative freedom from
interference by the usual diluents and
excipients in amounts for in excess of
their
normal
occurance
in
pharmaceutical preparations. The
results are given in table (3)
Table (3) Determination of 20µg of mesna in the presence of excipients and other
substancess
Interfering substances
Amount of
interferents
taken/mg
1
1
10
40
20
10
30
30
40
40
Na2- EDTA
Benzyl alcohol
Chlorobutanol
Lactose
Microcrystalline cellulose
Calcium phosphate
Corn starch
Povidone
Magnesium stearate
Hydroxylpropyl methyl
cellulose
Poly ethylene glycol
20
Titanrium dioxide
10
*Average of six determinations.
Amount found/µg
RSD %
Method A
Method B
Method A
method B
19.8
19.5
19.4
19.5
18.9
19.4
19.8
18.9
19.4
19.5
18.9
19.6
19.3
19.6
19.5
19.5
19.6
19.5
19.4
19.3
0.78
0.88
0.91
0.71
0.64
0.81
0.78
0.79
0.91
0.93
0.68
0.64
0.93
0.77
0.79
0.77
0.68
0.64
0.93
0.91
19.8
19.4
19.5
19.5
0.91
0.88
0.64
0.68
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3-4 Analytical applications
claim for pharmaceutical preparations
and the recovery results of real water
samples
was
higher
than
98%,indicating
that
successfully
applicability of the proposed methods.
Table(4),(5) gives the results of the
assay and reveals that there is close
agreement between the results obtained
by the proposed methods and the lable
Table (4): Determination of mesna in pharmaceutical formulations.
Pharmaceutical
Label amount
Found* (mg)
formulation
(mg)
Method A
Method B
Mesnan tablet(NDI) 400
402
401.3
Mesnan
100
99.7
99.9
ampoule(NDI)
*Mean value of six determinations.
Table(5): Determination of mesna in spikedwater samples
Water samples
Mesna(mg/ml)
% Recovery(n=6)
Taken
Found
Method
Method
Method
Method
A
B
A
B
Drinking water
0.1
0.101
101
101
0.101
98
99
1.0
0.98
98.66
98
0.99
1.5
1.48
1.47
Industrial waste water
0.1
0.1
0.1
100
100
1.0
1.02
102
101
1.01
100
1.5
1.5
100.6
1.51
*Mean of ten determinations
extraction. To the best of our
knowledge, this is the first report of the
reaction between mesna and excess of
sodium hypochlorite(NaOH) and then
indirectly determined the drug after the
residual hypochlorite bleaches the dyes
which absorb at different wavelength
maxima.
4- Conclusions
An
indirect
spectrophotometric
methods were established for the
analysis of mesna in pure form
,pharmaceuticals and water samples
.The proposed methods are simple
,rapid ,sensitive ,accurate ,selective,
and no need of heating or solvent
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15.A.Clezio
and
F.Orlando,
Quim.Nova, 2002,25; 387-391
16.
N.Kunio and S.Shigetaka, “,
Japan analyst, 1982,31; 724-726
17- ICH Harmonized tripartite
guideline prepared within the
international conference on the
harmonization of technical
requirements for the registration
of pharmaceuticals for
human
use (ICH) Q2B, validation of
analytical procedure: methodology.
ICH, London
1996
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